Variable Clinical Presentation of Glycogen Storage Disease Type IV: from Severe Hepatosplenomegaly to Cardiac Insufficiency

Letter to the Editor

Variable clinical presentation of glycogen storage disease type IV: from severe hepatosplenomegaly to cardiac insufficiency. Some discrepancies in genetic and biochemical abnormalities

Edyta Szymańska1, Sylwia Szymańska2, Grażyna Truszkowska3, Elżbieta Ciara4, Maciej Pronicki2, Yoon S. Shin5, Teodor Podskarbi6, Alina Kępka7, Mateusz Śpiewak8, Rafał Płoski9, Zofia T. Bilińska10, Dariusz Rokicki1

1Department of Pediatrics, Nutrition and Metabolic Disorders, the Children’s Corresponding author: Memorial Health Institute, Warsaw, Poland Dariusz Rokicki MD, PhD 2Department of Pathology, the Children’s Memorial Health Institute, Warsaw, Poland Department of Pediatrics, 3Department of Medical Biology, Molecular Biology Laboratory, Institute Nutrition and of Cardiology, Warsaw, Poland Metabolic Disorders 4Department of Medical Genetics, the Children’s Memorial Health Institute, Warsaw, Children’s Memorial Poland Health Institute 5University Children’s Hospital and Molecular Genetics and Metabolism Laboratory, Al. Dzieci Polskich 20 Munich, Germany 04-730 Warsaw, Poland 6Molecular Genetics and Metabolism Laboratory, Munich, Germany Phone: +48 22 815 75 45 7Department of Biochemistry, Radioimmunology and Experimental Medicine, E-mail: [email protected] the Children’s Memorial Health Institute, Warsaw, Poland 8Cardiac Magnetic Resonance Unit, Institute of Cardiology, Warsaw, Poland 9Department of Medical Genetics, Centre of Biostructure, Medical University of Warsaw, Warsaw, Poland 10Unit for Screening Studies in Inherited Cardiovascular Diseases, Institute of Cardiology, Warsaw, Poland

Submitted: 19 February 2017 Accepted: 26 June 2017

Glycogen storage disease (GSD) type IV (Andersen’s disease, amy- lopectinosis, polyglucosan body disease) is a rare inherited disorder of carbohydrate metabolism. The disease is caused by autosomal recessive mutations in the GBE1 gene (OMIM 607839), which leads to glycogen branching enzyme (GBE) deficiency. This is a critical enzyme in the pro- duction of both muscle and liver glycogen. Due to its decreased activity, abnormal long branched molecules of low solubility are formed. These deposits lead to glycogen precipitation in the liver, and subsequently build up in the body tissue, especially the heart and liver. The severity of the disease varies according to the amount of enzyme produced. In adults, the activity of GBE is higher and symptoms do not appear until later in life [1]. Therefore, clinical manifestations of GSD IV consist of different sub- types with variable ages of onset, severity, and clinical features. The fatal perinatal neuromuscular subtype, which presents in utero with decreased fetal movements, polyhydramnios, and fetal hydrops, and congenital neuromuscular subtype beginning in the newborn period with profound hypotonia, respiratory distress, and dilated cardiomyopathy, both result in death in the neonatal period. The classic progressive hepatic subtype presents with rapidly developing failure to thrive following birth. Clinical manifestations include hepatomegaly, liver dysfunction, E, Szymańska, S. Szymańska, G. Truszkowska, E. Ciara, M. Pronicki, Y.S. Shin, T. Podskarbi, A. Kępka, M. Śpiewak, R. Płoski, Z.T. Bilińska, D. Rokicki and progressive liver cirrhosis, as well as hypoto- ous parents of Polish origin. The pregnancy was nia, and cardiomyopathy. In this case, death due not complicated, but slow physical development to liver failure usually occurs by the age of 5 years was observed from the 28th gestation week. La- unless liver transplantation (LTx) is performed. bor was complicated with oligoamnios, and pelvic The non-progressive hepatic subtype presents longitudinal lie. Apgar scores were 6–7–8 respec- with hepatomegaly, liver dysfunction, myopathy, tively. Psychomotor development delay and poor and hypotonia. However, individuals with this type body mass gain were observed from her perina- usually do not show progression of liver disease, tal period. The child presented with mild hypo- and may not even have cardiac, skeletal muscle, or tonia, dysplasia of the hip, and pes equine. From neurologic involvement. The childhood neuromus- 5 weeks of age elevated liver enzymes, cholesta- cular subtype, which is the rarest one, has most sis (bilirubin: total 32.7 μmol/l, direct 14.3 μmol/l) variable course. Its onset ranges from the second and increased cholesterol and triglyceride levels life decade with a mild disease course to a more were found and persisted. At the age of 9 months severe, progressive course resulting in death in the girl remained microsomic (body mass – 2.0 SD, the third decade [2, 3]. height – 3.48 SD), her psycho-motor development The disease diagnosis and management is thus was markedly delayed, and liver and spleen were multidisciplinary, and should include specialists in enlarged. Lysosomal storage diseases suspected metabolic disorders, hepatology, neurology, and due to very high serum chitotriosidase activity nutrition, as well as in medical or biological ge- (5000 nmol/ml/h, ref. < 150) were excluded by en- netics. zymatic testing. During subsequent months por- We report three cases of Polish patients with tal hypertension, muscle hypotonia of lower limbs, mutations in the GBE1 gene with various clinical and hypertrophic cardiomyopathy developed. The courses of their disease each. girl required endoscopic intervention three times Patient 1: The girl was born in 2006 as a former due to the massive bleeding from esophagal vari- 2.4 kg term infant to healthy, non-consanguine- ces performed at the age of 19 months.

A B

C D

Figure 1. Histological findings in patients 1 and 2.A – Patient 1: explanted liver showing cirrhosis and extensive amylopectin deposits. PAS stain. Original magnification 200×.B – Patient 1: Autopsy, heart muscle with numerous intrasarcoplasmic PAS-positive deposits. PAS stain. Original magnification 200×.C – Patient 1: Autopsy, brain nervous tissue with numerous globular PAS-positive deposits located in the white matter. Original magnification 400×. D – Patient 2: Core needle liver biopsy. Intrahepatocytic amylopectin deposits. PAS stain. Original magnification 200×

238 Arch Med Sci 1, January / 2018 Variable clinical presentation of glycogen storage disease type IV: from severe hepatosplenomegaly to cardiac insufficiency. Some discrepancies in genetic and biochemical abnormalities

Liver transplantation was performed at the Autopsy revealed: (1) heart muscle and peri- age of 22 months. Complete micronodular cir- cardial involvement with extensive intracytoplas- rhosis, sparse diffuse lymphocytic infiltration mic PAS-positive deposits in cardiomyocytes (Fig- and presence of large or granular PAS-positive ure 1 B) and subpericardial macrophages, and intracytoplasmic inclusions located in numerous (2) diffuse central nervous system damage caused hepatocytes were found in the liver explant (Fig- by widespread deposition of numerous small glob- ure 1 A). In routine hematoxylin and eosin stain ular PAS-positive deposits. Their location included inclusions displayed basophilic or amphophilic the cerebral and cerebellar cortex as well as white staining characteristics. Transmission electron matter (Figure 1 C). microscopy of the liver revealed aggregates con- Patient 2: The boy was born in 1999 as a for- sistent with amylopectin deposition. Enzymatic mer 4.14 kg term infant of a 3rd uncomplicated study showed brancher deficiency both in leuko- pregnancy to healthy, non-consanguineous par- cytes and in the liver but normal activity in fibro- ents of Polish origin. The labor was also not com- blasts (Table I). plicated and the Apgar score was 10. His psycho- After LTx the girl’s hepatic function improved, motor development was good. At 9 months of age her mental development was good, but her mo- mild hepatomegaly and hypertransaminasemia tor skills were still greatly delayed; she was hypo- (ASP 319 U/l, ALT 263 U/l) were found on a rou- tonic, presented with muscle atrophy and did not tine health maintenance visit. A complete gastro- walk. Her body mass was at the 50th percentile, logic and metabolic work-up was performed at and height at the 3rd percentile. At 6.5 years of the age of 13 months and revealed deficiency of age, the girl was admitted to the hospital due to branching enzyme (Table I), which indicated the symptoms of sepsis with pulmonary abscess and GSD type IV. Brancher activity in leukocytes of the severe breathing difficulties. She died after a few patient’s parents was at the lower limit of the nor- weeks of intensive medical care. mal range. Liver core needle biopsy performed at

Table I. Activities of enzymes involved in synthesis and degradation of glycogen and glycogen amount in morphotic elements of the blood in three patients with molecularly confirmed GSD type IV

Activity* Patient 1 Patient 2 Patient 3

Liver:

Brancher (ref. 0.3–3.0) 0.0 0.0 –

Debrancher (ref. 0.17–0.72) – 0.21 –

Fibroblasts:

Brancher (ref. 0.5–3.0) 1.7 0.16 0.30 (c 1.73; 2.31) (c 1.5; 0.98) (c 0.99) Leukocytes:

Brancher (ref. 0.22–0.81) 0.11 0.04 0.20 (M 0.28; F 0.20) (M 0.20; F 0.16; B 0.16) Debrancher (ref. 26.8–105):

Phosphorylase a with AMP (ref. 1.0–3.2) – 1.2 2.1

Phosphorylase a without AMP (ref. 0.46–2.2) – – 1.24

–

FBP-1,6-ase (ref. 0.89–15.9) – 4.6 –

Erythrocytes –

Glycogen (ref. 5.7–135) – 16.7 112

Phosphorylase b kinase (ref. 8.6–45.9)

Activity* Patient 1 Patient 2 Patient 3

Liver

M – mother, F – father, B – brother, c – control, FBP-1,6-ase – fructose-1,6-biphosphatase; *Units brancher – µmol Pi/min/mg protein, debrancher in liver (leukocytes) – µmol glucose/mg protein/hour (nmol glucose/mg protein/h), phosphorylase a (with, without ± AMP) – µmol Pi/h/mg protein, glycogen – µg/g hemoglobin, phosphorylase b kinase – µmol Pi/min/g hemoglobin. FBP-1,6-ase – µmol/min/mg protein. **Normal ranges of brancher in Laboratory in Munich: leukocytes 0.22–0.81; fibroblasts 0.5–3.0; liver 0.3–3.

Arch Med Sci 1, January / 2018 239 E, Szymańska, S. Szymańska, G. Truszkowska, E. Ciara, M. Pronicki, Y.S. Shin, T. Podskarbi, A. Kępka, M. Śpiewak, R. Płoski, Z.T. Bilińska, D. Rokicki the age 26 months showed moderate fibrosis hav- 192 cm height, and he had no congestive signs. ing no cirrhotic stage of nodular transformation. On 12-lead standard electrocardiogram sinus Intrahepatocytic inclusions had the same morpho- rhythm was present, short PR of 116 ms, left atrial logical and staining characteristics as in patient 1 enlargement and ST-T segment changes in lateral (Figure 1 D). leads (Figure 2). Cardiac magnetic resonance im- Treatment included a high protein diet with re- aging (MRI) study confirmed bidimensional Dop- striction of non-utilizable sugars and regular food pler echocardiography findings of the nondilated intake. The boy has been following routine health left ventricle with mildly depressed left ventricle maintenance visits every 3 months at the be- function of 51%. Moderate mitral insufficiency ginning, and every 6 months afterwards. Further was also present with mitral insufficiency frac- follow-up revealed mild microalbuminuria of 311 tion of 23%. Late gadolinium enhancement imag- mg/day (ref. < 30 mg/day). Thus, nephroprotective es showed hyperintense areas in midmyocardial treatment with an ACE inhibitor was commenced. basal and partly medial segments of the inter- There has been no further progression of renal ventricular septum (Figure 3). Repeated 24-hour complication since that time. Holter electrocardiogram showed multiform ven- The boy is now 17 years old. He has been in tricular arrhythmia up to 2500 of single ventric- good health. His liver is of normal size, and there ular extrasystoles and complex 10–20 base pairs is no evidence of liver function abnormalities. The and infrequent short nsVT episodes. The patient patient has experienced normal growth and pu- was diagnosed with left ventricular dysfunction berty with normal life activities. associated with mitral insufficiency and compli- Patient 3: The 24-year old student experienced cated with multiform ventricular arrhythmia. Liver the first episode of heart failure at the age of enzymes and creatine kinase serum concentra- 5 months and was then treated with catechol- tions were within normal limits. The patient was amines. Since then, impaired exercise tolerance, treated pharmacologically (sotalol, candesartan) nonspecific chest pain and frequent palpitations on and did not agree to ICD implantation. During exercise had been observed. At the age of 21 years 3 years of follow-up the patient’s left ventricu- the patient underwent an electrophysiologic study lar ejection fraction decreased to 35%, and the (due to PR of 70 ms on standard 12-lead electro- patient started having paroxysmal atrial fibrilla- cardiogram) that ruled out Wolff-Parkinson-White tion. Neurological examination was normal. The syndrome, and radiofrequency (RF) ablation of family history revealed RF ablation of ventricular frequent ventricular ectopy in the right ventric- arrhythmia in the right ventricular outflow tract ular outflow tract was performed. At the age of at the age of 49 years in the mother. At present, 22 years he was referred to the Institute of Cardi- aged 50 years she is mildly hypertensive with- ology because of multifocal ventricular arrhythmia out any symptoms; a 12-lead electrocardiogram to make a diagnosis. He complained of recurrent, revealed sinus rhythm of 61/min, and short PR longer episodes of palpitations and decreased ex- of 96 ms; 2D Doppler echocardiogram was nor- ercise tolerance. His blood pressure (BP) was of mal. The proband’s father, aged 47 years, is free 110/60 mm Hg, heart rate 64/min, weight 104 kg, of symptoms, obese, treated for diabetes type 2

Figure 2. 12-lead ECG of patient 3 which shows sinus rhythm, short PR, left atrial enlargement and ST-T segment changes in lateral leads

240 Arch Med Sci 1, January / 2018 Variable clinical presentation of glycogen storage disease type IV: from severe hepatosplenomegaly to cardiac insufficiency. Some discrepancies in genetic and biochemical abnormalities

A B

Figure 3. Late gadolinium enhancement images of patient 3 show hyperintense areas in midmyocardial basal (A) and partly medial segments of the interventricular septum (B) and hypercholesterolemia, with normal electro- ic coding or splicing variants with frequency no cardiographic and echocardiographic study. Whole greater than 0.01 in ExAC, ESP6500 and POL400 exome sequencing (WES) was performed and databases in 174 genes from cardiological panel. did not reveal any pathogenic/likely pathogen- The patient carried the heterozygous ACTN2

A B

C D

Figure 4. Integrative Genomics Viewer view of GBE1 pathogenic variants in patient 1 (A, B) and patient 3 (C, D) by whole-exome sequencing

Arch Med Sci 1, January / 2018 241 E, Szymańska, S. Szymańska, G. Truszkowska, E. Ciara, M. Pronicki, Y.S. Shin, T. Podskarbi, A. Kępka, M. Śpiewak, R. Płoski, Z.T. Bilińska, D. Rokicki

NM_001103.3:c.2497G>A: (p.Ala833Thr) variant GBE1 gene in the trans position (Figure 4, Table inherited from his mother classified in ClinVar II). Serum branching enzyme activity was below as a variant of uncertain significance. Additional the lower normal range, indicating the diagnosis potentially pathogenic variants were found in the of GSD type IV (Table I). Chitotriosidase activity in

Table II. Details of molecular variants identified in GBE1 in three patients with GSD type IV

Variable Patient 1 Patient 2 Patient 3

Mutant allele Mutant allele Mutant allele Mutant allele Mutant allele Mutant allele 1 2 1 2 1 2 Chromosome/ 3/rs1042498 3/– 3 3 3/ 3/ rs rs137852888 rs754607405 RefSeq cDNA NM_000158.3 NM_000158.3 NM_000158.3 NM_000158.3

Nucleotide c.263G>A c.1621A>T IVS5 + 2t>c c.1570C>T c.2056T>C change Exon 2 13 12 16

IGV reads 12/28 6/11 ND ND 14/49 27/68

RefSeq protein NP_000149 NP_000149 NP_000149 NP_000149

Amino acid p.(Cys88Tyr) p.(Asn541Tyr) p.(His528Arg) p.(Arg524*) p.(Tyr686His) change Mutation:

Type Missense Missense Nonsense Missense

Stage Heterozygous Heterozygous Heterozygous Heterozygous Heterozygous Heterozygous

Status Novel Novel Known (PMID Novel 8613547, 15452297) Parental Paternal Maternal Maternal Paternal origin Pathogenicity prediction: CADD* 1.23 18.49 41.0 21.9

PolyPhen2 Benign Probably – Probably damaging damaging Mutation High Neutral – High Assessor (functional) (functional) LRT Deleterious Deleterious Deleterious Deleterious

Mutation- Disease Disease Disease Disease Taster causing causing causing causing automatic SIFT Tolerated Damaging

MAF data**:

ExAC 60,706 0.000014 0 0.00001685 0.00000817

ESP 6500 0 0 0.00016 0

EUR 1000 0 0 0 0

POL 400 0 0 0 0

Depth of WES 27 11 66 49

*Higher scores are more deleterious, **MAF – minor allele frequency; ExAC – exome aggregation consortium (http://exac.broadinstitute. org); EUR 1000 – 1000 genomes project (http://www.1000genomes.org/); ESP 6500 – exome sequencing project (http://evs.gs.washington. edu/EVS); POL400 – project of 400 exomes from Polish individuals with unrelated diseases (unpublished).CADD – Combined Annotation Dependent Depletion (http://cadd.gs.washington.edu/home); PolyPhen-2 – Polymorphism Phenotyping v2 (http://genetics.bwh.harvard. edu/pph2/index.shtml); MutationAssessor (http://mutationassessor.org); LRT – Likelihood Ratio Test (http://www.genetics.wustl.edu/ jflab/lrt_query.html).

242 Arch Med Sci 1, January / 2018 Variable clinical presentation of glycogen storage disease type IV: from severe hepatosplenomegaly to cardiac insufficiency. Some discrepancies in genetic and biochemical abnormalities

Table III. Clinical and laboratory characteristics at diagnosis of 3 patients with mutations in GBE1 gene

Patient Clinical presentation Laboratory abnormalities

1 Severe course: hepato-splenomegaly, Elevated serum: neurological involvement, – Liver enzymes liver failure with cholestasis – Chitotriosidase (5000 nmol/ml · h) – Lipids – Lactate cholestasis, coagulative impairment 2 Hepatomegaly without enlarged spleen Hypertransaminasemia and no impairment of liver function Elevate CK, chitotriosidase (317–613 nmol/ml/h) 3 Cardiological presentation: dilated No specific laboratory abnormalities cardiomyopathy and mild arrhythmia serum was assessed and found to be in the nor- In patients 1 and 3 mutations in GBE1 were mal range (29 nmol/ml/h, ref. < 150). There were identified by WES and in patient 2 by Sanger se- neither class I clinical indications for endomyocar- quencing. dial biopsy, nor for liver or muscle biopsy. Branch- The sequencing run for patient 1’s sample ing enzyme activity was assessed in the patient’s achieved 52 579 854 reads with a mean value of fibroblasts (Munich, Y. Shin, T. Podskarbi), and it 32.08. Above 64% and 87% of the target region was somewhat lower than that of the control cul- was covered min. 20 and 10 times, respectively. ture (Table II). The sequencing run for patient 3 sample achieved Table III presents clinical characteristics and 72 905 031 reads with a mean value of 39.63. laboratory studies for all 3 patients at diagnosis. Above 73% and 91% of the target region was cov- Activity of glycogen-branching enzyme was ered min. 20 and 10 times, respectively. measured using the spectrophotometric method In patients 1 and 3 two compound heterozy- described by Brown [4, 5]. Briefly, as a result of gous nucleotide substitutions in the GBE1 gene activity of glycogen-branching enzymes (amylo- (NM_000158.3; NP_000149) inherited in trans (1,4-1,6) transglycosylase; EC 2.4.1.18) and were identified by WES in probands and confirmed phosphorylase, synthesis of glycogen from glu- by bidirectional Sanger sequencing in probands cose-1-phosphate occurs. Resulting from the re- and in their parents (Figure 4, Table II). In patient 1 action inorganic phosphate was measured spec- c.263G>A:(p.Cys88Tyr) of paternal origin and trophotometrically (spectrophotometer Cary 1-E, c.1621A>T:(p.Asn541Tyr) of maternal origin and Varian, Mulgrave, Australia). Its concentration is in patient 3 c.1570C>T:(p.Arg524*) of maternal or- proportional to the enzymatic activity. Enzymat- igin and c.2056T>C:(p.Tyr686His) of paternal ori- ic assays performed in the Laboratory in Munich gin were identified. All variants were heterozygous (Y. Sinn, T. Podskarbi) used C14 UDP glucose in- and inherited from unaffected parents. Both of the stead of phosphorylase [6, 7]. variants in patient 1 are apparently novel missense DNA was extracted from the peripheral blood mutations. Cys88Tyr is known as a polymorphism and used for massively parallel exome sequenc- and Tyr686His is predicted to be pathogenic. Sub- ing. WES Library preparation was performed using stitution c.263G>A (p.Cys88Tyr) affects a weakly Nextera Rapid Capture Exome kits (Illumina). The conserved amino acid in glycoside hydrolase, fam- samples were run on 1/4 lane each on HiSeq 1500 ily 13, N-terminal carbohydrate-binding module using 2 × 75 bp paired-end reads. Bioinformatics 48 (CBM48), but a large physicochemical differ- analysis was performed as previously described ence between Cys and Tyr (Grantham score 194) [8]. The detected variants were annotated using is observed. Substitution c.1621A>T (p.Asn541Tyr) Annovar and converted to Microsoft Access format concerns a highly conserved amino acid in glycosyl data for final manual analyses. Alignments were hydrolase, family 13, subfamily, catalytic domain viewed with the Integrative Genomics Viewer. and likewise a large physicochemical difference Variants identified in the GBE1 gene with next between Asn and Tyr (Grantham score 143). generation sequencing (NGS) were followed up in In addition, the variants in patient 1 were pre- probands and their parents with Sanger sequenc- dicted by in silico analysis (Table II) as probably ing using a 3500xL Genetic Analyzer (Life Technol- damaging enzyme activity, which suggests that ogies, Carlsbad, CA, USA) and BigDye Terminator they are disease-causing genetic factors. The v3.1 Cycle Sequencing Kit (Life Technologies) ac- c.263G>A substitution is referenced on dbSNP cording to the manufacturer’s instructions. The (rs1042498) and recorded on ExAC databases (Eu- results were analyzed with Variant Reporter 1.1 ropeans: 1/37098 alleles (0.0027%); ALL: 1/69262 software (Life Technologies). (0.0014%)). The c.1621A>T substitution is absent A list of primers specific to each GBE1 variant is on dbSNP, ESP6500 and ExAC databases (as of available on request. 14.04.2016).

Arch Med Sci 1, January / 2018 243 E, Szymańska, S. Szymańska, G. Truszkowska, E. Ciara, M. Pronicki, Y.S. Shin, T. Podskarbi, A. Kępka, M. Śpiewak, R. Płoski, Z.T. Bilińska, D. Rokicki

In patient 2, sequencing of GBE1 revealed com- variant was predicted as deleterious by six in sili- pound heterozygous IVS5 + 2t>c/H528R geno- co algorithms (CADD, PolyPhen2, Mutation Asses- type, which is deemed to be associated with the sor, LRT, Mutation Taster, SIFT). The c.2056T>C:(p. mild form of GSD IV [9]. Tyr686His) variant was reported in the ExAC data- The c.1570C>T:(p.Arg524*) variant, found in base (1/120668 [0.000008287]) and is absent in patient 3, was previously described in 2 patients. the ESP6500 database. One of those patients had a classic hepatic pre- A large variety of clinical manifestations of GSD sentation carrying Arg524* and Phe257Leu GBE1 type IV have been reported, from early onset he- variants [PMID 8613547] [9] and the other patient patic presentation with a probable end result is with neuromuscular presentation carried Arg524* cirrhosis and death within 5 years, to mild liver and His628Arg variants [15452297] [10]. The disease without progression in people reaching second variant in patient 3 (SM) c.2056T>C:(p. adulthood without liver transplantation [11]. Tyr686His) is a novel variant affecting a conserved Genotype-phenotype correlations remain un- amino acid in the C-terminal amylase-like barrel clear but are emerging. Fatal perinatal neuro- domain of GBE1. The c.2056T>C:(p.Tyr686His) muscular subtype and congenital neuromuscular subtype of GSD IV are usually caused by 2 null mutations, classic hepatic subtype by compound heterozygous for a null and missense mutation and adult polyglucosan body disease by homozygous or compound heterozygous missense mutations. Milder forms of GSD IV are usually associated with 2 missense mutations [10762170, 15452297] and more severe forms of GSD IV with at least one null mutation or large deletion [17662246, 15520786, 8613547, 15452297, 12913206, 17662246]. However there are excep- tions such as – NM_000158.3:c.2003delA found in an APBD patient [23034915] and homozygous missense mutations p.H545R in fatal perinatal cases [15452297] are also observed. To our best knowledge to date, 63 mutations in the GBE1 gene have been reported. With our 4 novel missense mutations this number increases to 67, of which 34 (51%) are missense mutations, 7 (10%) nonsense, 7 (10%) splice-site mutations, 12 (18%) frameshift mutations, and 6 (9%) large (above > 10 bp) deletions/insertions. Most missense mutations are found in the catalytic domain of GBE, and cluster in exons 5–8 and 12–13 (Figure 5). In some patients only one mutation in GBE1 was identified [8613547, 15452297, 10545044, 20058079]. A recent article by Akman et al. showed deep intronic mutation in a repetitive sequence of intron 15 which was originally missed in whole-exome sequence data [25665141]. We report three cases of Polish patients with mutations in the GBE1 gene, each presenting with a different clinical course of the disease. Patient 1 was found with elevated chitotriosidase activity, hepato-splenomegaly and liver dysfunction, which led to the initial diagnosis of Nie- mann-Pick disease type A/B. This is not the first case report of GSD IV being a rare cause of elevated chitotriosidase activity and diagnosed through genetic testing [12]. Since chitotriosidase activity is known to be directly associated with acute or Figure 5. Missense mutations found in catalytic do- chronic inflammation and macrophage activation main of GBE, and clustered in exons 5–8 and 12–13 [13], its elevated serum activity in patients with

244 Arch Med Sci 1, January / 2018 Variable clinical presentation of glycogen storage disease type IV: from severe hepatosplenomegaly to cardiac insufficiency. Some discrepancies in genetic and biochemical abnormalities

GSD IV may be due to lipid accumulation, which with KD have demonstrated an improvement of triggers peroxidation in hepatocytes and activa- cardiomyopathy in echocardiography [19]. Since tion of liver resident macrophages and Kupffer the mechanism (storage of pathologic substance cells and secretion of proinflammatory cytokines. in the heart muscle) of heart enlargement in both Therefore, diagnosis of GBE deficiency should be GSD types is similar, there is a possibility that KD considered in the differential diagnosis in patients may also have a positive impact on cardiomyopa- with chronic liver disease and elevated chitotrio- thy and myopathy in GSD IV. sidase activity, once lysosomal storage diseases A similar case to our patient was published for have been excluded. The patient required LTx to the first time in 1988 by Greene et al. [20]. Almost counteract chronic liver failure, which is a com- 10 years later, in 1996, McConkie-Rosell et al. pre- mon indication in the classical form of GSD IV, but sented the clinical spectrum of non-progressive unfortunately died due to LTx complications. In liver form of GSD IV in 4 patients, and long-term this case histopathologic examination of the liv- follow-up of the oldest identified patients (ages er and postmortem heart and brain biopsy were 13 and 20 years) [21]. These case reports have crucial for diagnosis. One of the variants found proved that type IV glycogen storage disease is in the GBE1 gene is only possibly pathogenic but more heterogeneous and may possibly be more presence of a deletion in GBE1 on a second allele, common than previously suspected. unrevealed by WES study, cannot be excluded. Patient 3 demonstrated the least known form Since GSD IV is a rare disease, the data on its of GSD IV: isolated cardiac involvement, but with natural history after Ltx are limited. In classical progressive myocardial dysfunction. He did not infantile liver form Ltx remains the only effective fulfill the criteria of neuromuscular subtype of treatment for progressive liver failure. According GSD IV, a “polyglucosan body disease”, because to the largest review so far, fewer than 20 patients he has not presented with either neurological with this type of GSD have been transplanted [14]. or skeletal muscle symptoms, at least until now. This analysis of 17 patients who had undergone There was no clinical or laboratory evidence clear- Ltx due to liver cirrhosis by 2008 demonstrated ly pointing to such a diagnosis for years and the that the majority of patients (12/17) survived, 2 of diagnosis was established by chance, through ap- them required a second LTx, and most complica- plication of NGS [22]. Chitotriosidase activity was tions were related to sepsis or hepatic thrombosis also within normal values and branching enzyme not associated with cardiomyopathy. Skeletal my- activity was only slightly decreased. A causal link opathy and cardiomyopathy did not develop after between the GBE1 defect and the cardiac involve- LTx in most cases. A few other single case reports ment in patient 3 seems very probable although published so far have confirmed the efficacy of not confirmed at the functional level. In the liter- LTx for patients with classic hepatic form, but the ature Aksu et al. reported the first hypertrophic data on rare, atypical forms are still not available cardiomyopathy case due to type IV GSD in the [15–17]. literature [23]. Based on the phenotype of case 3, The initial manifestation of GSD IV in patient 2 a detailed search of desmosomal (PKP2, DSP, was slight hepatomegaly with elevated hepatic DSG2, DSC2, JUP), and nondesmosomal genes transaminases and CK activity during the first year (TMEM43, LMNA, PLN, CTNNA3) linked to arrhyth- of life. In this case, enzyme (brancher) activity in mogenic cardiomyopathy (ARC) was performed, the liver was 0, but in leukocytes was not, which yielding no results [24, 25]. Although the majority is compatible with the mild variant liver form (Ta- of cases of ARC are transmitted in an autosomal ble I). The liver often gives a much lower enzyme dominant way, a few of them, such as cardiocu- activity value, partly due to the bad condition and taneous syndromes like Naxos disease (related partly due to the complicated method of Pi pro- to JUP) and Carvajal disease with dominant left duction using phosphorylase a. ventricular, related to DSP, are transmitted in an After the diagnosis was established, proper autosomal recessive way. Our patient was free of medical management with a high protein diet any cutaneous symptoms. Of interest, both the and carbohydrate restriction was commenced. Al- patient and his mother had short PR on 12-lead though data supporting such nutritional manage- electrocardiogram. In the literature, short PR/ ment are limited, single studies have shown that preexcitation is characteristic of glycogenoses, patients with GSD IV may benefit from a high pro- especially in Danon disease and PRKAG2 disease, tein diet similarly to those with type III and IX [18]. but also is found in Anderson-Fabry disease, mito- This particularly applies to muscular forms (pa- chondrial disease and most commonly is associat- tients with cardiomyopathy) since for infants with ed with hypertrophic cardiomyopathy [15]. classic hepatic form Ltx is the only possible treat- In the proband, a compound heterozygote ment. There are no data on the efficacy of keto- of GBE1 variant carrier, short PR was associat- genic diet (KD) in GSD IV, but single case reports of ed with myocardial thinning due to fibrosis, and patients with GSD III and cardiomyopathy treated in his mother, a single GBE1 variant carrier with

Arch Med Sci 1, January / 2018 245 E, Szymańska, S. Szymańska, G. Truszkowska, E. Ciara, M. Pronicki, Y.S. Shin, T. Podskarbi, A. Kępka, M. Śpiewak, R. Płoski, Z.T. Bilińska, D. Rokicki normal myocardial function. Storage/infiltrative Since the natural course of this multi-systemic disorders with short PR may be transmitted in au- metabolic disease is still unknown, further studies tosomal dominant, recessive, X-linked and matri- on this issue are necessary. linear fashion [12]. No LT has been necessary so In conclusion, GSD IV should be suspected not far. However, since long-term prognosis of cardiac only in patients with liver cirrhosis and liver failure. involvement in GSD IV (or in the carriers of the The disease should also be considered in the differ- GBE1 mutation) has not been known yet, further ential diagnosis in individuals with hepato-spleno- studies on its genetic and clinical features should megaly and elevated chitotriosidase activity once be performed in order to understand the genuine lysosomal storage diseases have been excluded, as pathogenesis and course of the disease. well as in patients with isolated and progressive These 3 case reports of Polish patients with myocardial dysfunction associated with features mutation in the GBE1 gene and various courses of preexcitation on ECG. Biochemical, and partic- of their disease confirm the clinical heterogeneity ularly, histological evaluation plays a crucial role in of this disorder. Our observation also shows re- the diagnosis of GSD IV, especially in cases with markable difficulties in establishing a conclusive unclear molecular findings, since polyglucosan diagnostic decision even in the “WES era”, as in bodies are highly diagnostic, microscopic features patient 3. Pathogenicity of his Tyr686His variant for glycogen branching enzyme deficiency. is not proven by in vitro/in vivo studies and only heterozygous status of GSD type IV cannot be ex- Acknowledgments cluded on enzymatic verification. Functional test- We acknowledge Prof. dr hab. n.med. Ewa Pron- ing of the Tyr686His pathogenicity is not available icka for substantive supervision and an invaluable and probably only a future study on polyglucosan contribution to the concept and preparation of the bodies in heart bioptate will appear conclusive. manuscript. When it comes to LTx, progression to liver This study was partially financed by CMHI proj- failure in patients with other than classical liv- ect S136/13 and 235/15. er forms is hard to predict. Therefore, close fol- This research was enabled on the basis of low-up including both clinical and laboratory (liver a grant received from the Danone Medical Trading enzymes, coagulation and other parameters of B.V. which encompasses Nutricia Metabolics. liver function) monitoring should be provided. However, the presence of normal liver size and Conflict of interest function assessed by laboratory examinations in adults with GSD IV renders progression to liver The authors declare no conflict of interest. failure rather unlikely, although periodic assess- ment including serum liver enzymes and coagula- References tion parameters may be helpful. In children more 1. Li SC, Chen CM, Goldstein JL, et al. 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246 Arch Med Sci 1, January / 2018 Variable clinical presentation of glycogen storage disease type IV: from severe hepatosplenomegaly to cardiac insufficiency. Some discrepancies in genetic and biochemical abnormalities

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